Project Description The broad objective of this proposal is to understand the mechanism of genetic recombination and its relationship to chromosome maintenance in both Bacteria and Eukarya. Our approach is to reconstitute increasingly more complex reactions that recapitulate steps of the recombination process, using purified proteins from Escherichia coli and from Saccharomyces cerevisiae, and using sophisticated biochemical and biophysical methods of analysis, which include single-molecule imaging. The specific aims of this proposal are divided along two broad aims. The first set of aims describes our ongoing efforts to mechanistically understand the steps of E. coli recombination in vitro. This aim includes studies on the function of RecN protein; the mechanism of the DNA homology search by RecA; and the processing of double Holliday junctions. The second major aim is to continue to biochemically reconstitute the processes that comprise recombination in S. cerevisiae. This aim includes studies of the function of the chromatin remodelers; the coupling of DNA resection to DNA pairing; and the mechanism of the DNA homology search by Rad51.